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United States Department of Agriculture

Agricultural Research Service

Title: Stable Ethanol Production by Pet Operon-Containing Escherichia Coli Strainslacking Lactate Dehydrogenase and Pyruvate-Formate Lyase

item Wyckoff, Herbert
item Hespell, Robert
item Dien, Bruce
item Bothast, Rodney

Submitted to: Journal of Industrial Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: August 9, 1996
Publication Date: N/A

Technical Abstract: In the last decade, a major goal of biofuels research has been to metabolically engineer microorganisms for high ethanol yield from a variety of agricultural biomasses. E. coli strains with the PET operon (pyruvate decarboxylase and alcohol dehydrogenase) make high levels of ethanol, but require antibiotics for genetic stability. To overcome this major commercial developmental problem, we used mutational complementation by transforming the PET operon plasmid pLOI295 into E. coli K12/FMJ39, which cannot grow anaerobically due to lactate dehydrogenase and pyruvate-formate lyase mutations. The resulting strain, FBR1, grew anaerobically and was genetically stable without the addition of ampicillin due to conditional lethality imposed by plasmid loss under anaerobic conditions. In fleaker fermentors with 10% substrate, strain FBR1 made 3.9% ethanol from glucose, 3.5% ethanol from arabinose, and xylose utilizing revertants of FBR1 made 3.8% ethanol from xylose. Transforming the PET operon plasmid pLOI297 into strain FMJ39 yielded strain FBR2, which also grew anaerobically, was extremely stable without the addition of tetracycline (Tc), and produced 4.4% ethanol from glucose. In a chemostat, after 12 days/48 generations (23 with and 25 without Tc), strain FBR2 did not lose Tc resistance or ability to produce high levels of ethanol. Genetic improvements to strains FBR1 and FBR2, along with ethanol fermentations using sugar hydrolysates from corn fiber, are being conducted.

Last Modified: 4/22/2015